Abstract
Epidermal growth factor (EGF) and transforming growth factor alpha bind to a common receptor at the cell surface. Both the affinity and the tyrosine protein kinase activity of the receptor are regulated by exogenous factors, such as platelet-derived growth factor. A protein kinase C-dependent (Ca2+/phospholipid-dependent enzyme) and independent regulatory mechanism have been described. The protein kinase C-dependent mechanism results in the inhibition of the affinity and tyrosine kinase activity of the EGF receptor. We describe in this report an alternative mechanism of regulation of the receptor that is mediated by sphingosine. Treatment of WI-38 human fetal lung fibroblasts with 5 microM sphingosine for 2 min at 37 degrees C caused a marked increase in the affinity of the EGF receptor. Similar results were obtained when isolated plasma membranes prepared from these cells were incubated with sphingosine. A stimulation of the EGF receptor tyrosine protein kinase activity was also observed after sphingosine-treatment of plasma membranes. Sphingosine caused a decrease in the Km for ATP and an increase in the Vmax for the tyrosine phosphorylation of a synthetic peptide substrate. Control experiments demonstrated that these actions of sphingosine were not secondary to the inhibition of protein kinase C. These data indicate that sphingosine causes the functional conversion of the EGF receptor into an activated state that expresses both a high affinity for EGF and an increased tyrosine kinase activity. We conclude that sphingosine is a bioactive molecule in human fibroblasts.
Highlights
D when the concentration of ATP was changed
Functional Activity of the EGF Receptor Is Regulated by Two Alternative Pathways-In this report we demonstrate that sphingosine causes an increase in the affinity and tyrosine protein kinase activity of the EGF receptor
As sphingosine and lysosphingolipids have been reported to be potent inhibitors of protein kinase C (Hannun et al, 1986;Hannun andBell, 1987)it is possible that theactivation of the functional activity of theEGF receptor caused by sphingosine is secondary to the inhibition of protein kinase C
Summary
Sphingosine Stimulates EGF Receptor TyrosineKinase Activity-Incubation of membranes prepared from WI-38 fibroblasts with sphingosine caused an increase in the autophosphorylation of the EGFreceptor (Fig. 1).A similar result was observedwhen the membranes were incubated with EGF. In addition to stimulating the autophosphorylation of the EGF receptor, sphingosine increastehde phosphorylation of an exogenous synthetic peptide substrate (Table I). Analysis of the kinetic parameters of the tyrosine phosphorylation demonstrated that both sphingosine and EGF caused a decrease in the K,, for substrate (ATP)and an increase in the VmaXcompared with control EGF receptors (Table I). This result is in marked contrast to the effect of protein kinase C which caused a decrease in the V,, of the phosphorylation reaction (Table I). Sphingosine caused a marked increase in EGF receptor tyrosine protein kinase activity, no effect of Nacetylsphingosine was observed (data not shown).
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